Fig. 4. The DT1 element of the Emx2 enhancer contains adjacent Tcf- and Smad-binding sites (A) Nucleotide sequence of DT1. The canonical Tcf- and Smad-binding sites (underlined) are indicated. For mutational analysis, the underlined nucleotides were replaced by GC and C, respectively. (B) The binding capacity of the Emx2 enhancer was tested in electrophoretic mobility shift assays with recombinant Lef1 and Smad1MH1 protein. Lef1 and Smad1 bind to an Emx2 enhancer oligonucleotide (lanes 2 and 3, respectively). Binding is potentiated in the presence of both proteins (lane 4) and an additional slower migrating, Lef1/Smad1 complex is observed. Complex formation is competed by a molar excess (100x) of the Emx2 enhancer oligonucleotide but not by an oligonucleotide containing mutations in the Tcf- and Smad-binding sites (lanes 5-8).
